This invention relates to substrates for use in immobilizing biomolecules. More particularly, the invention relates to substrates (e.g. glass slides) having a coating of polylysine covalently attached to a silane layer coating the slide, wherein the polylysine compound has functional NH2 groups which can be used to either directly immobilize biomolecules (e.g. DNA molecules), covalently, or non-covalently; or be used for attachment of linkers for subsequent immobilization of biomolecules. Even more particularly, the invention relates to specific prescribed addition of ethanalomine to the polylysine thereby forming a mixture which dramatically enhances the effectiveness of the polylysine for immobilizing DNA. Among other applications, the polylysine coated substrates can be used in the preparation of high density arrays for performing hybridization assays.
Polylysine coatings have been utilized for years in the modification of glass and plastic in order to create a biocompatible surface for cell attachment. Additionally, polylysine has been utilized as a coating that will immobilize biomolecules such as DNA for use in hybridization assays. The DNA is held to the surface by electrostatic forces between the positively charged side chain of the polylysine and the net negative charge of the DNA molecule. Polylysine has several advantages over other popular DNA attachment chemistries such as gamma-aminopropyltriethoxysilane (GAPS). For one, polylysine functionalizes a substrate surface with a greater concentration of amino groups than GAPS, which in turn results in greater retention of DNA. Second, the polylysine molecule, when attached to a substrate, is flexible and extends from the surface, and therefore is more suitable for the binding and hybridization of large polymeric molecules such as DNA.
Unfortunately, Polylysine is not very stable on glass substrates. As a polycation, it electrostatically interacts with the negatively charged surface of the glass. However, a common step in any DNA hybridization assay is a blocking step, which is used to block any non-specific binding of the sample DNA probes. For example, succinic anhydride, a commonly used blocking agent confers a net negative charge to the polylysine. This effectively blocks the non-specific DNA sample binding, but also has the effect of weakening the electrostatic attachment of the polylysine to the glass surface.
The present invention presents a surface coating method and attachment chemistry which takes advantage of the benefits of polylysine while obviating the problem of its inherent instability by covalently attaching the polylysine to the glass by means of a silane compound.
The present invention discloses a coating that when applied to a substrate surface, allows for the effective immobilization of biomolecules. The coating comprises a silane layer covalently attached to the substrate and a polycation covalently attached to the silane layer. The polycationic layer possesses functionalities that will immobilize biomolecules.
The present invention further discloses mixing the polycation with an auxiliary nucleophile that has the effect of controlling the amount of covalent attachment of the polycation to the substrate surface. In a specific example, an optimal ratio range of the polylysine (polycation) and ethanolamine (auxiliary nucleophile) mixture has been identified.
The present invention further discloses a high density nucleic acid array whereby different known genetic sequences are attached at a plurality of locations to a substrate having a silane layer covalently attached to the substrate surface and a polycation layer covalently attached to the silane layer. The gene sequences are electrostatically attached to the polycation layer. Again, the effectiveness of the polycationic layer to immobilize the DNA may be modulated by selective addition of an auxiliary nucleophile diluent such as ethanalomine.
The present invention also discloses a method of preparing a substrate for biomolecular immobilization comprising the steps of: providing a substrate with a surface; attaching a silane layer to the surface; mixing a functionalizes polymer with a diluent to form a mixture; and, attaching the mixture to the silane coated substrate. A further embodiment provides for the attachment of biomolecules (e.g., DNA, RNA, proteins, antigens, ligands, haptens, etc.) to the substrate via the functionality of the cationic polymer.
Still another embodiment calls for chemical modification of the polycationic layer to create reactive esters at the surface. These reactive esters have the ability to covalently attach various ligands.